Method for inactivating viruses using electron beams

ABSTRACT

The invention relates to a method for inactivating viruses, characterized in that an immunogenic composition or vaccine comprising at least one virus is irradiated with electron beams, said immunogenic composition or vaccine comprising at least one virus (i) being liquid, in particular being a suspension and (ii) comprising at least one viral immunogen, wherein the antigen structure is preferably substantially retained.

The invention relates to a method for inactivating viruses,characterised in that an immunogenic composition or vaccine comprisingat least one virus is irradiated with electron beams, said immunogeniccomposition or vaccine comprising at least one virus (i) being liquid,in particular being a suspension and (ii) comprising at least one viralimmunogen.

Through the use of vaccines, many infectious diseases in human andveterinary medicine can be successfully fought. Nevertheless, there isstill a major need for vaccine technologies which provide effective andlong-lasting protection against infections but which are without risksto the vaccinated individual. This is significant in the preparation ofso-called dead vaccines: to inactivate viruses toxic chemicals such asformaldehyde are used which must then be removed again from the vaccineby complex processes. In veterinary medicine, formaldehyde-inactivatedvaccines make up the majority of all vaccines and in human medicine theyare used, for example, against TBEV, influenza, poliomyelitis orhepatitis A. The use of formaldehyde leads to a chemical alteration(cross-linking) of the viral antigens. This in turn results in anattenuated vaccine efficacy, which must be compensated for by anincreased amount of infectious starting material and effect enhancers(adjuvants). A technique to circumvent these problems is a clear unmetneed in the vaccine industry.

Studies have shown up to 30%-80% of antigens which are important for asuccessful vaccination are destroyed by formaldehyde (Amanna et al.,Nature Medicine, 18, 2012). These problems are known to the vaccineindustry and alternatives are sought. For instance, experiments havebeen conducted with UV rays, raised temperatures, gamma-rays orperoxides. To date, none of these techniques have progressed beyond theexperimental laboratory phase.

It has also been reported that Salmonella populations can be reduced byusing high-energy electron beams (U.S. Pat. No. 8,173,139B1). However,the extent to which the structure of antigens is impaired was notdescribed therein. Moreover, Salmonella are living organisms with theirown metabolism. The applicability of electron beams to viruses which,outside of their host cells, have no inherent metabolism, couldtherefore not be assumed.

In the present invention it has now been found, surprisingly, thatviruses could be inactivated with electron beams without destroyingviral structural proteins. Surprisingly, therefore, irradiation withelectron beams is suitable, particularly for preparing inactivated viralwhole particle vaccines.

The viruses are presumably inactivated by destroying the nucleic acidwherein the virus structure, and in particular the antigen structure, isundamaged or barely damaged. The results using the example of the pigpathogenic PRRSV virus are shown in Examples 1 and 2 and FIGS. 1 to 3.In this case, a liquid, namely a suspension of these viruses in bufferedaqueous solution, was treated with electron beams. This suspension isimmunogenic, and is suitable as a vaccine against PRRSV in pigs.

The results with the influenza A virus are shown in Example 3 and FIGS.4 and 5. In this case, a liquid, namely a suspension of these viruses inbuffered aqueous solution, was treated with electron beams. Thissuspension is immunogenic, and is suitable as a vaccine againstinfluenza A.

In one embodiment, the present invention relates to a method forinactivating viruses, characterized in that an immunogenic compositionor vaccine comprising at least one virus is irradiated with electronbeams, said immunogenic composition or vaccine comprising at least onevirus (i) being liquid, in particular being a suspension, and (ii)comprising at least one viral immunogen.

In a preferred embodiment, the at least one virus is active prior toirradiation and/or the immunogenic composition or vaccine comprisesactive viruses. In particular, the concentration of active viruses inthe liquid composition or vaccine prior to irradiation, as measured byTCID50 value (50% tissue culture infective dose) per ml of liquid, is atleast 10⁴, 10⁵ or 10⁶ per ml.

In another embodiment, the invention relates to a method forinactivating viruses, characterized in that a composition comprising atleast one virus is irradiated with electron beams, said compositioncomprising at least one virus (i) being liquid, in particular being asuspension, and/or (ii) being an immunogenic composition.

The method can be applied to any viruses, particularly enveloped ornon-enveloped viruses.

In a preferred embodiment, it is suitable for enveloped viruses, sinceenvelope proteins are suitable antigens for a vaccine reaction. Theantigen structure remains intact using the method according to theinvention, while the viruses themselves are inactivated (see FIGS. 1, 2and 3). Surprisingly therefore, using the method according to theinvention, in particular an inactivated viral whole particle vaccine canbe prepared for enveloped viruses.

In another preferred embodiment, an inactivated viral whole particlevaccine can be prepared for non-enveloped viruses. A suitable antigenfor a vaccine reaction in non-enveloped viruses is particularly a capsidprotein of the virus.

The following viruses, for example, may be irradiated in accordance withthe invention:

enveloped viruses, for example:

-   -   double-stranded DNA viruses, for example:    -   poxvirus (causes smallpox);    -   herpes viruses (e.g. herpes simplex (HSV) (causes herpes        labialis or genitalis); varizella zoster virus (VZV) (causes        chickenpox and shingles) Epstein-Barr virus (EBV) (causes        glandular fever) cytomegalovirus (CMV) (causes cytomegaly)    -   human herpes virus 6 7 (causes roseola)    -   human herpes virus 8 (HHV 8) (causes Kaposi's sarcoma)    -   hepadnaviruses, for example:    -   hepatitis B virus (causes hepatitis B)    -   (+)-strand RNA viruses, for example:    -   flaviviruses, for example:    -   hepatitis C virus (causes hepatitis C)    -   togaviruses, for example:    -   rubella virus (causes rubella)    -   coronaviruses (causes gastrointestinal infections, SARS)    -   (−)-strand RNA viruses, for example:    -   orthomyxoviruses, for example:    -   influenza viruses A, B or C (causes influenza) paramyxoviruses,        for example:    -   parainfluenza viruses (causes parainfluenza)    -   measles virus (causes measles)    -   mumps virus (causes mumps)    -   respiratory sincytical virus (RSV)    -   pneumoviridae, for example the genera:    -   pneumovirus, metapneumovirus    -   rhabdoviruses, for example:    -   rabies virus (causes rabies)    -   retroviruses, for example:    -   human immunodeficiency virus (causes AIDS)    -   HTLV (causes leukemia)

Non-enveloped viruses, for example:

-   -   double-stranded DNA viruses, for example:        -   adenoviruses (causes colds, common colds)        -   papovaviruses (causes warts)    -   single-stranded DNA viruses, for example:        -   parvoviruses, for example:            -   parvovirus B19 (causes fifth disease)    -   double-stranded RNA viruses, for example:        -   rotaviruses (diarrhea)    -   (+)-strand RNA viruses, for example:        -   picornaviruses, for example:            -   polio virus (causes poliomyelitis)            -   coxsackieviruses            -   echoviruses            -   hepatitis A virus (causes hepatitis A)            -   rhinoviruses (causes colds, common colds)        -   caliciviruses (causes diarrhea)

In a preferred embodiment, the at least one virus is thus selected from:

(i) an enveloped virus or non-enveloped virus, in particular anenveloped virus, and/or

(ii) a dsDNA virus, dsRNA virus, ssRNA virus or ssDNA virus, and/or

(iii) a human pathogenic and/or animal pathogenic virus.

In a preferred embodiment, the animal is a mammal, selected inparticular from pigs, cows, horses, dogs, cats and sheep.

In a more preferred embodiment, the at least one virus is selected froma human pathogenic and/or animal pathogenic enveloped dsRNA virus,enveloped ss(−)RNA virus or enveloped ss(+)RNA virus.

It has been shown in the examples that the PRRS virus (porcinerespiratory and reproductive failure syndrome virus), a single-stranded,positive strand RNA virus of the family Arteriviridae which affectspigs, was inactivated by the method according to the invention such thatthe virus structure and antigen structure remained largely intact.Therefore, an inactivated viral whole particle vaccine against the PRRSvirus could be prepared.

In an even more preferred embodiment, the at least one virus is selectedfrom a human pathogenic and/or animal pathogenic ss(+)RNA virus, veryparticularly preferably from a virus of the Arterividae family, and evenmore preferably the at least one virus is a porcine reproductive andrespiratory syndrome virus (PRRS virus).

In a further preferred embodiment, the at least one virus is selectedfrom an echo virus, HIV virus, rotavirus, pseudorabies virus,parvovirus, porcine parvovirus, H5N1 virus, H1N1 virus, Epstein-Barrvirus, mumps virus, influenza A and B virus, the TBEV virus, the IPVvirus and the hepatitis A virus.

In a further preferred embodiment, the at least one virus is selectedfrom an animal pathogenic virus, the influenza A and B virus, the TBEVvirus, the IPV virus and the hepatitis A virus. Particular preference isgiven to the influenza A and B virus.

Using the method according to the invention, it is possible to irradiatean immunogenic composition or vaccine comprising one (1) virus, as shownin the examples with PRRSV. It is also possible, however, that theimmunogenic composition or vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10or more different viruses. This may be helpful, for example, forpreparing combination vaccines. These two or more different viruses canbe variants of the same virus species or viruses of different species,families or genera. In a preferred embodiment, therefore, theimmunogenic composition or vaccine comprises (i) one virus or (ii) twoor more different viruses. However, it is also possible to prepare acombination vaccine in which two or more viruses are irradiatedindividually, and are only combined after irradiation.

In the examples, a suspension of PRRSV viruses was irradiated withelectrons which were accelerated to less than 300 keV, for example, to150 keV (see FIGS. 1 to 5). Such electrons are accelerated at lowenergy.

In a preferred embodiment, the method according to the invention ischaracterized in that the electron beams are accelerated at low energyor moderate energy, preferably accelerated with an acceleration energyof between 150 keV and 700 keV, more preferably of between 200 keV and500 keV, even more preferably of between 250 keV and 400 keV.

In this case, it is possible to operate under standard atmosphericpressure or essentially under standard atmospheric pressure and theelectron beams can therefore be applied preferably essentially understandard atmospheric pressure. Essentially standard atmospheric pressureis understood to mean 1 bar+/−0.1 bar. The standard atmospheric pressurecan be present here, for example, as atmospheric oxygen, nitrogen, orcarbon dioxide gas.

As shown in the examples, the PRRSV virus could be inactivated using adose of 50 kGy, 100 kGy or 200 kGy. Furthermore, it could be shown thatthe influenza A virus in suspension is completely inactivated at a doseof 200 kGy.

It has been found that a dose of at least 50 kGy is advantageous inorder to achieve, as far as possible, complete inactivation of theviruses.

In a preferred embodiment, the method according to the invention istherefore characterized in that the immunogenic composition or vaccinecomprising at least one virus is irradiated with an electron beam doseof at least 50 kGy, at least 60 kGy, at least 70 kGy, at least 80 kGy,at least 90 kGy, at least 100 kGy, at least 110 kGy, at least 120 kGy,at least 130 kGy, at least 140 kGy, at least 150 kGy, at least 160 kGy,at least 170 kGy, at least 180 kGy, at least 190 kGy, at least 200 kGyor at least 250 kGy.

Furthermore, it has been found that a dose of 300 kGy or less isadvantageous in order to avoid potential damage to the composition.

In a preferred embodiment, the method according to the invention istherefore characterized in that the immunogenic composition or vaccinecomprising at least one virus is irradiated with an electron beam doseof at most 300 kGy, at most 250 kGy, at most 200 kGy, at most 190 kGy,at most 180 kGy, at most 170 kGy, at most 160 kGy, at most 150 kGy, atmost 140 kGy, at most 130 kGy, at most 120 kGy, at most 110 kGy, at most100 kGy, at most 90 kGy, at most 80 kGy, at most 70 kGy or at most 60kGy.

In a preferred embodiment, the electron beam dose is therefore in therange of 50 kGy to 300 kGy. For example, the immunogenic composition orvaccine comprising at least one virus may be irradiated at an electronbeam dose of 50 kGy, 60 kGy, 70 kGy, 80 kGy, 90 kGy, 100 kGy, 110 kGy,120 kGy, 130 kGy, 140 kgy, 150 kGy, 160 kGy, 170 kGy, 180 kGy, 190 kGy,200 kGy, 210 kGy, 220 kGy, 230 kGy, 230 kGy, 240 kGy, 250 kGy, 260 kGy,270 kGy, 280 kGy, 290 kGy or 300 kGy.

In a preferred embodiment, the method according to the invention istherefore characterized in that the immunogenic composition or vaccinecomprising at least one virus is irradiated with an electron beam dosein the range of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, furtherpreferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, evenmore preferably 50 kGy to 110 kGy.

In another embodiment of the method according to the invention, the atleast one virus is irradiated with an electron beam dose of 1 to 300kGy, more preferably with an electron beam dose of 1 to 150 kGy, evenmore preferably with an electron beam dose of 10 to 120 kGy, mostpreferably with an electron beam dose of 15 to 110 kGy.

In example 1, it was shown that an inactivation by 2.5 logarithmic stepscould be achieved by irradiation with electrons by means of the methodaccording to the invention (FIG. 1). The inactivation was determined bymeasuring the TCID50 value.

In a preferred embodiment, the method according to the invention istherefore characterized in that the activity of the at least one virusafter irradiation, preferably measured as a TCID50 value (50% tissueculture infective dose), is less than 5%, preferably less than 1%, morepreferably less than 0.1% of the activity prior to irradiation, evenmore preferably that no activity of the at least one virus is stilldetectable after irradiation. The TCID50 value can be determined asdescribed in Example 2. The person skilled in the art will thereforeselect a suitable tissue culture for a particular virus. For the PRRSVvirus in Example 2, the TCID50 value was determined in Marc-145 cells.For the influenza A virus in Example 3, the TCID50 value was determinedin MDCK cells.

It could be shown in the examples for the enveloped PRRSV virus that theantigen structure was substantially retained under the statedconditions: the binding of a polyclonal serum directed against thenon-inactivated virus to the at least one virus of the irradiatedcomposition corresponded to more than 90% of the binding to thenon-inactivated virus (see FIG. 1). The antigen structure of influenzaviruses which had been treated with an electron beam dose of 200 kGy wasalso substantially retained (FIG. 5). The binding of a polyclonal serumfrom an influenza A infected human to the virus irradiated with 200 kGycorresponded to about 80% of the binding to the non-inactivated virus.

In a further embodiment, the method according to the invention istherefore characterized in that the at least one virus is an envelopedvirus, and that the antigen structure of the viruses of the immunogeniccomposition or vaccine was substantially retained after irradiation.

In another further embodiment, the method according to the invention istherefore characterized in that the at least one virus is anon-enveloped virus, and that the antigen structure of the viruses ofthe immunogenic composition or vaccine was substantially retained afterirradiation.

Antigens are substances, especially proteins, capable of bindingspecifically to antibodies. To act as antigens, the antigens or theepitopes present therein must be chemically and structurally intact. Thepreservation of the two-dimensional and/or three-dimensional structureof the antigens or the epitopes present therein is frequently necessaryfor binding to the antibodies. It has now been found, surprisingly, thaton irradiation with electron beams the antigen structure of theirradiated viruses is largely retained and thus the irradiated liquidcomposition may be used as a vaccine to induce a specific immuneresponse in a human or animal, particularly a mammal.

An epitope is a region of an antigen to which an antibody specificallybinds.

In a particularly preferred embodiment, the binding of a polyclonalserum directed against the non-inactivated virus to the at least onevirus of the irradiated immunogenic composition or vaccine is at least40%, preferably at least 70%, more preferably at least 80%, even morepreferably at least 90% of the binding of the polyclonal serum to the atleast one virus of the immunogenic composition or vaccine prior toirradiation.

The binding of the polyclonal serum to the at least one virus of theimmunogenic composition or vaccine is preferably determined in this caseby ELISA. The determination of the binding by ELISA is preferablycarried out as set out in the examples.

ELISA (enzyme-linked immunosorbent assay) refers to an antibody-baseddetection method which has been well known for decades to those skilledin the art. Using ELISA, substances such as proteins can be detected.What is exploited here is the property of specific antibodies which bindto the substance, the antigen, to be detected. An antibody is firstlabelled with an enzyme. The reaction catalyzed by the reporter enzymeserves as evidence for the presence of the antigen. A substrate isconverted by the enzyme and the reaction product can then be detected,for example by measuring the absorption or chemiluminescence.

In a further embodiment, the method according to the invention istherefore characterized in that the at least one virus is an envelopedvirus and that the antigen structure of the viruses of the compositionis substantially retained after irradiation.

In another further embodiment, the method according to the invention istherefore characterized in that the at least one virus is anon-enveloped virus and that the antigen structure of the viruses of thecomposition is substantially retained after irradiation.

In another embodiment, the binding of a polyclonal serum directedagainst the non-inactivated virus to the at least one virus of theirradiated composition is at least 40%, preferably at least 70%, morepreferably at least 80%, even more preferably at least 90% of thebinding of the polyclonal serum to the at least one virus of thecomposition prior to irradiation

The binding of the polyclonal serum to the at least one virus of thecomposition is preferably determined in this case by ELISA. Thedetermination of the binding by ELISA is preferably carried out in thiscase as set out in the examples.

It is furthermore advantageous for the preparation of immunogeniccompositions and vaccines for non-enveloped and enveloped viruses if thevirus structure of the viruses is substantially retained afterirradiation. This applies particularly if a whole-body vaccine,particularly inactivated whole-body vaccine, is desired. Whole-bodyvaccines have the advantage that they comprise the various antigens ofthe virus and can therefore trigger a comprehensive immune response. Inthe examples, it could be shown for an enveloped virus that the bindingof an antibody directed against an antigen of the virus, which isinaccessible to the antibody in the case of intact envelopes of thisvirus, namely the N-protein of PRRSV, is essentially the same in theirradiated composition and the negative control, while in theperoxide-treated composition an increase in the binding of over 400% wasobserved.

A whole-body vaccine or whole particle vaccine is a vaccine comprising avirus in which the virus particle structure is substantially retained.

An inactivated whole-body vaccine or whole particle vaccine is a killedvaccine comprising a virus in which the virus structure is substantiallyretained.

A killed vaccine comprises inactivated or killed viruses or bacteria orconstituents of viruses, or bacteria or toxins. These can no longerreplicate in the body.

An immunogen according to the present invention is an antigen which iscapable of eliciting an immune response due to its immunogenicity.

An immunogenic composition is a composition which is capable ofeliciting an immune response in a human or mammal. An immunogeniccomposition comprises at least one immunogen. In a preferred embodiment,an immunogenic composition is suitable for administration to a human oran animal, and is thus prepared for administration to an animal orhuman. The composition therefore preferably contains no substances whichare not approved or are unsuitable for administration to a human or ananimal, in particular, no carcinogenic, highly allergenic or toxicsubstances.

A vaccine according to the present invention comprises at least oneantigen and/or immunogen which, due to its immunogenicity, is capable ofeliciting an immune response and is prepared for administration to ananimal or human. A vaccine is suitable for administration to a human oranimal. A vaccine therefore preferably contains no substances which arenot approved or are unsuitable for administration to a human or animal,in particular, no carcinogenic, highly allergenic or toxic substances.

A vaccine directed against a virus preferably provides protectionagainst a viral infection or ameliorates this disease. A vaccinedirected against a virus is therefore preferably suitable for theprevention and/or treatment, for example, the alleviation of a viralinfection or disease in a human or animal, in particular a mammal.

In a preferred embodiment, the method according to the invention istherefore characterized in that the virus structure of the viruses issubstantially retained after irradiation.

In a preferred embodiment, the method according to the invention istherefore characterized in that the at least one virus is an envelopedvirus, and that the virus structure of the viruses is substantiallyretained after irradiation.

In a further preferred embodiment, the method according to the inventionis therefore characterized in that the at least one virus is anon-enveloped virus and that the virus structure of the viruses issubstantially retained after irradiation.

In the context of the present invention, a virus structure issubstantially retained if the binding of an antibody directed against anantigen of the virus, which is inaccessible to the antibody in the caseof an intact envelope, to the at least one virus of the composition,particularly immunogenic composition or vaccine, after irradiation isless than 400%, preferably less than 200%, more preferably less than150%, even more preferably less than 120% of the binding of thisantibody to the at least one virus of the composition, particularlyimmunogenic composition or vaccine, prior to irradiation. This ispreferably determined by ELISA, as shown in the examples. It has beenshown in FIG. 3, surprisingly, that on irradiation of a liquidimmunogenic composition comprising PRRSV viruses, the capsid (N−)protein is not accessible using an antibody against this antigen in anELISA.

In a further embodiment, therefore, the present invention also relatesto the use of a method according to the invention for preparing aninactivated viral whole particle vaccine.

In the method according to the invention electron beams are used. In theindustrial generation of electrons—after power-up—the accelerationvoltage between the cathode and anode applied in the emission of theelectrons to their energy content is given in electron volt eV. Thehigher this is, the deeper the electrons provided with more energy canpenetrate into matter. The radiation power is the product of beamcurrent (amount of electrons generated) and accelerationvoltage—specified in kilowatts (kW). At high radiation powers, theenergy dose required for triggering chemical reactions by irradiationcan be generated and applied in very short times (fractions of asecond). Overall, electron beams, despite lower penetration, areparticularly advantageous in throughput processes of high amounts ofproduct: physicochemical processes can be initiated and implemented in atechnically readily adjustable manner. A major advantage is that theradiation process can be switched on or off virtually at the push of abutton (unlike gamma rays for example).

Suitable devices for generating electron beams and devices for carryingout the method are known from the prior art (A. Heger: Technologie derStrahlenchemie von Polymeren [Technology of radiation chemistry ofpolymers], Carl Hanser Verlag Munich Vienna, 1990. ISBN 3-446-15630-5;Chapter 2: Allgemeine Grunglagen [General principles]; pp. 25-39 andChapter 4: Industrielle Bestrahlungsanlagen [Industrial irradiationplants]; pp. 69-149; DE 196 389 25 A1).

Electron beams are also advantageous compared to gamma radiation: sinceconventional gamma sources often only deliver ca. 2 to 6 kGy per hour,higher doses can only be achieved over long time courses, whereaselectron beams reach this in a few seconds (high dose rates) and aretherefore substantially more productive.

In conventional gamma irradiation in air in commercial servicefacilities, a long-term concomitant oxidative effect (ozone in air or OHfree radicals from water) on the treated material or the reactionprocess occurs, which often does not, but occasionally does bring aboutproduct alterations and product outcomes in the sense desired. In thecase of short dose impact by electron beam systems, most of which arealso designed to operate under inert atmosphere, such side effects areruled out over a longer impact time.

The method according to the invention is preferably carried out using adevice for generating electron beams which is operated continuously orrapidly pulsed.

In a further preferred embodiment, the method according to the inventionis carried out using a device for generating electron beams whichprovides electrons according to the cold or hot cathode principle.

In a further preferred embodiment, the method according to the inventionis carried out using a device for generating electron beams which isembodied as an axial emitter (scanner) or linear broadband emitter.

In a further preferred embodiment of the method according to theinvention, the electrons are applied to the composition after dischargethrough an emission window of the evacuated generating chamber of thedevice. In this case, the composition, in particular an immunogeniccomposition or vaccine, is preferably in a container.

In a further preferred embodiment of the method according to theinvention, the composition, in particular an immunogenic composition orvaccine, is incorporated statically in the device, or is continuouslytransported through the electron beam.

Furthermore, the dose rate (beam current/time) can be adjusted. In thiscase, it is generally to be born in mind—with respect to the desiredfinal applied dose—that a high beam current requires little irradiationtime and low beam current requires a long irradiation time. The doserate is adjusted by a person skilled in the art by considering, forexample, the flow rate of the liquid medium in the split tube, as wellas the type of emitter.

For example, a dose can be applied in the range of 10 to 300 kGy,particularly 50 to 300 kGy, over a time period of up to 1000 seconds;lower doses of 1 to 25 kGy can be applied preferably over a time periodof 0.1 to 100 seconds. Doses above 50 kGy, which are preferred,typically require an application time of 10 seconds to 1000 seconds.Therefore, the application time is preferably 10 seconds to 1000seconds.

In a further preferred embodiment of the method according to theinvention, the dose rate is therefore in the range of 1 kGy/0.1 secondsto 150 kGy/1000 seconds.

In a further preferred embodiment of the method according to theinvention, the irradiation time is therefore in the range of 0.1 secondsto 1000 seconds, preferably between 1 second and 100 seconds.

During the irradiation, a temperature increase typically takes place. Toavoid denaturing processes, it is therefore advantageous if thetemperature rises only slightly.

In a further preferred embodiment of the method according to theinvention, the temperature of the composition, in particular animmunogenic composition or vaccine, prior to irradiation is thereforebetween 1° C. and 40° C., preferably between 5° C. and 37° C., morepreferably between 10° C. and 32° C., even more preferably between 15°C. and 30° C.

In another embodiment, it is possible to carry out the irradiation attemperatures of the composition prior to irradiation of less than 1° C.,for example, of frozen compositions. In this case, the composition afterirradiation can also have a temperature of less than 1° C., or thetemperature of the composition after irradiation can be 1° C. or more.

In a further preferred embodiment of the method according to theinvention, the temperature increase of the composition, in particular animmunogenic composition or vaccine, after irradiation compared to beforeirradiation is between 1° C. and 15° C., preferably between 2° C. and10° C.

In a further preferred embodiment of the method according to theinvention, the temperature of the composition, in particular animmunogenic composition or vaccine, after irradiation is thereforebetween 2° C. and 41° C., preferably between 6° C. and 38° C., morepreferably between 11° C. and 33° C., even more preferably between 16°C. and 31° C.

The composition, in particular an immunogenic composition or vaccinecomprising at least one virus is liquid. In this case, such a liquid canpreferably be in the form of a suspension of viruses in an aqueoussolution, as in the examples; however, it may also be a suspension ofhigher density.

In a further preferred embodiment of the method according to theinvention, therefore, the density of the composition, in particular animmunogenic composition or vaccine, is between 0.9 and 2 g/cm³,preferably between 1.0 and 1.8 g/cm³.

In a further preferred embodiment of the method according to theinvention, therefore, the composition, in particular an immunogeniccomposition or vaccine comprising at least one virus, comprises a liquidsuspension comprising water, preferably a suspension of the at least onevirus in an aqueous solution, wherein the aqueous solution particularlypreferably comprises one or more buffer substances. The aqueous bufferedsolution may be PBS for example. The pH of such a solution is preferablyin the range of pH 5.5 to 8.5, more preferably in the range of pH 6.5 to8.0.

It is possible using the method according to the invention to irradiatean otherwise finished vaccine which already contains suitableauxiliaries and/or adjuvants.

In a further embodiment of the method according to the invention,therefore, the immunogenic composition or vaccine comprises (a) one ormore adjuvants and/or (b) pharmaceutically acceptable excipients and/orauxiliaries and/or (c) one or more further immunogens.

In a further preferred embodiment of the method according to theinvention, therefore, the immunogenic composition or vaccine consists ofat least one virus, in particular (1) one virus and

(a) one or more adjuvants, and/or

(b) pharmaceutically acceptable excipients and/or auxiliaries, such aswater or a suitable aqueous solution, particularly preferably comprisingone or more buffer substances,

and (c) optionally one or more further viruses or immunogens.

In a further embodiment of the method according to the invention,therefore, the composition is a vaccine comprising at least one viralimmunogen and optionally comprising (a) one or more adjuvants and/or (b)pharmaceutically acceptable excipients and/or auxiliaries and/or (c) oneor more further immunogens.

Suitable excipients and auxiliaries and/or further adjuvants are wellknown to those skilled in the art. Suitable adjuvants are those that aresufficient to enhance an immune response to an immunogen. Suitableadjuvants are, for example, aluminum salts such as aluminum phosphate oraluminum hydroxide, squalene mixtures (SAF-1), muramyl peptide, saponinderivatives, mycobacterium cell wall preparations, monophosphoryl lipidA, mycolic acid derivatives, nonionic block copolymer surfactants,Quil-A, cholera toxin B subunit, polyphosphazene and derivatives andimmunostimulating complexes (ISCOMs) such as those described inTakahashi et al. (1990) Nature 344:873-875.

Suitable excipients and auxiliaries are, for example, water or anaqueous solution suitable for administration, particularly preferablycomprising one or more buffer substances.

Suitable further immunogens are well known to those skilled in the artand particularly include

(a) organic substances, particularly proteins which may be glycosylatedor non-glycosylated, nucleic acids, toxins or sugar molecules,particularly saccharides optionally bound to a support, and

(b) a virus or a living organism, particularly a bacterium, wherein thevirus or living organism may be active or inactivated.

Suitable further immunogens are particularly those which induce animmune response to a pathogen or disease factor in the same animal orhuman as the at least one virus of the composition. For example, if theat least one virus is a human pathogenic virus, further immunogensshould be preferably selected such that they trigger an immune responseto a human pathogen and/or prevent or ameliorate a human disease.

In the case of lyophilized vaccines, stabilising agents, for example, apolyol such as sucrose or trehalose, may be added as excipients andauxiliaries.

As customary in all immunogenic compositions or vaccines, theimmunologically effective dose must be determined empirically. Factorsthat should be taken into account here are whether an immunogen shouldbe complexed with an adjuvant or carrier molecule or should becovalently bound thereto, the type of administration and the number ofimmunizing doses which should be given. Such factors are well known inthe field of vaccine development and a person skilled in this field canreadily determine these factors.

The at least one virus and optionally one or more further immunogens maybe present at different concentrations in immunogenic compositions orvaccines of the present invention. The minimum concentration in avaccine is typically one that is sufficient for its planned use forvaccination, wherein during the irradiation according to the methodaccording to the invention lower concentrations can also be used and ahigher concentration can then be adjusted in the finished vaccine orduring the irradiation according to the method according to theinvention higher concentrations can also be used and a lowerconcentration can then be adjusted in the finished vaccine. The maximumconcentration for the irradiation according to the method according tothe invention is typically one in which the at least one virus remainshomogeneously suspended during the irradiation and/or optionally one ormore further immunogens remain dissolved or homogeneously suspendedduring the irradiation. The maximum concentration in a vaccine istypically one at which the at least one inactivated virus remainshomogeneously suspended and/or optionally one or more further immunogensremain dissolved or homogeneously suspended.

The vaccines of the present invention may be used to protect or to treata human or an animal, particularly a mammal, by administration,particularly by systemic administration or administration via the mucousmembrane. The type of administration may be selected by those skilled inthe art and includes, for example, injection via the intramuscular,intraperitoneal, intradermal or subcutaneous route or the administrationvia the mucous membrane of the oral, respiratory or genitourinary tract.

The preparation of vaccines is described in general in Vaccine Design(“The subunit and adjuvant approach”, eds. Powell M. F. & Newman M J.)(1995) Plenum Press New York).

Alternatively, however, it is also possible in accordance with theinvention to initially irradiate a composition, particularly animmunogenic composition, comprising at least one virus and subsequentlyoptionally to add suitable auxiliaries and/or adjuvants. Furtherimmunogens for preparing combination vaccines can also be added.

In a further embodiment, the invention therefore relates to a method forpreparing a vaccine comprising at least one viral immunogen, inparticular a vaccine comprising a viral whole particle vaccine,characterized in that:

(a) the method according to the invention is carried out as describedabove,

(b1) one or more adjuvants are added to the composition, particularly animmunogenic composition, comprising at least one virus, and/or

(b2) one or more pharmaceutically acceptable excipients and/orauxiliaries are optionally added to the composition, particularly animmunogenic composition, comprising at least one virus, and/or

(b3) one or more further immunogens are optionally added to thecomposition, particularly an immunogenic composition, comprising atleast one virus,

wherein the steps (a) to (b3) are carried out in any sequence.

To be suitable for application to an animal or a human, and to ensuresecure transport and application at a defined dose, such a vaccine isusually sterile and filled into a suitable container. Such a containermay comprise multiple doses or single doses.

In a further preferred embodiment, the method according to the inventionfor preparing a vaccine is characterized in that the following furthersteps are carried out:

-   -   (c) sterilizing the immunogenic composition, and/or    -   (d) filling the immunogenic composition in a container,

wherein steps (a) to (d) may be carried out in any sequence, andfollowing steps (a) to (d) the vaccine is optionally dried, freeze-driedor frozen.

In a further embodiment of the method according to the invention,therefore, the composition comprising at least one virus is in addition

(c) sterilised, and/or

(d) filled in a container,

wherein steps (a) to (d) may be carried out in any sequence, and thenthe vaccine is optionally dried, freeze-dried or frozen.

The immunogenic compositions and vaccines obtained by the methodaccording to the invention are clearly superior to the compositions ofthe prior art since they do not comprise any residues of chemicalinactivating substances (such as formaldehyde) and/or and arecharacterized by the intact antigen structure of the virus withsimultaneous inactivation of the at least one virus. This isparticularly the case in a whole particle vaccine and/or killed vaccine.

The invention therefore further relates to an immunogenic composition orvaccine, preferably vaccine, particularly preferably comprising aninactivated viral whole particle vaccine, which can be prepared by themethod according to the invention.

In a further embodiment, the invention relates to an immunogeniccomposition or vaccine, preferably vaccine, comprising an inactivatedviral whole particle vaccine for an enveloped or non-enveloped virus,preferably enveloped virus, characterised in that

(a) the activity of the virus in the immunogenic composition or vaccineis less than 10%, preferably less than 1%, more preferably less than0.1% of the activity of the same number of non-inactivated viruses, and

(b) the antigen structure of the inactivated viruses in the immunogeniccomposition or vaccine is substantially the same compared to the samenumber of non-inactivated viruses.

In a preferred embodiment of an immunogenic composition or vaccineaccording to the invention, the virus structure of the inactivatedviruses is substantially the same compared to the same number ofnon-inactivated viruses.

In a further preferred embodiment, the immunogenic composition orvaccine according to the invention has been irradiated with an electronbeam as described above.

In a further preferred embodiment, the immunogenic composition orvaccine according to the invention has been irradiated with an electronbeam dose of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, furtherpreferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, evenmore preferably 50 to 110 kGy. This dose allows effective inactivationof a virus, wherein at the same time the virus structure and antibodystructure of an enveloped or non-enveloped virus, preferably envelopedvirus, is retained.

In a further preferred embodiment, no activity of the at least one virusis still detectable in the composition, in particular an immunogeniccomposition or vaccine. This is particularly important for a deadvaccine and/or inactivated viral whole body vaccine.

In a further embodiment, the invention relates to an immunogeniccomposition, preferably vaccine, according to the invention for use as avaccine, particularly for the prevention or treatment, particularlyamelioration of viral infections or disorders which are caused by thevirus.

In a further embodiment, the invention relates to the use of electronbeams for inactivating viruses in an immunogenic composition or vaccinecomprising at least one virus, said immunogenic composition or vaccine(i) being liquid, in particular being a suspension and (ii) comprisingat least one viral immunogen.

In a further embodiment, the invention relates to the use of electronbeams for inactivating viruses in liquid compositions, preferably liquidimmunogenic compositions or vaccines, more preferably vaccines.

In a further embodiment, the invention relates to the use of electronbeams for preparing an inactivated viral whole particle vaccine.

In a preferred embodiment of the use according to the invention, theelectron beams are accelerated at low energy or moderate energy,preferably accelerated with an acceleration energy of between 150 keVand 700 keV, more preferably of between 200 keV and 500 keV, even morepreferably of between 250 keV and 400 keV and/or are applied essentiallyunder standard atmospheric pressure, wherein the standard atmosphericpressure is present as atmospheric oxygen, nitrogen or carbon dioxidegas.

In a preferred embodiment of the use according to the invention, theelectron beam dose is in the range of 50 kGy to 300 kGy, preferably 50kGy to 200 kGy, further preferably 50 kGy to 150 kGy, more preferably 50kGy to 120 kGy, even more preferably 50 kGy to 110 kGy.

In a further embodiment, the invention relates to the use of a devicefor generating electron beams for inactivating viruses in liquidcompositions, preferably liquid immunogenic compositions, morepreferably vaccines, particularly liquid vaccines.

In a further embodiment, the invention relates to the use of a devicefor generating electron beams for preparing an inactivated viral wholeparticle vaccine.

In a preferred embodiment of the uses according to the invention, thedevice is suitable for emitting electron beams accelerated at low energyor moderate energy, preferably suitable for emitting electron beamsaccelerated at an acceleration energy of between 150 keV and 700 keV,more preferably suitable for emitting electron beams accelerated at anacceleration energy of between 200 keV and 500 keV, even more preferablysuitable for emitting electron beams accelerated at an accelerationenergy of between 250 keV and 400 keV.

In a further preferred embodiment of the uses according to theinvention, the device is suitable for applying electron beamsessentially under standard atmospheric pressure.

In a further preferred embodiment of the uses according to theinvention, the device is suitable for emitting an electron beam dose of50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, further preferably 50kGy to 150 kGy, more preferably 50 kGy to 120 kGy, even more preferably50 kGy to 110 kGy.

In a further preferred embodiment of the uses according to theinvention, the device is suitable for emitting an electron beam dose of1 to 300 kGy, more preferably an electron beam dose of 1 to 150 kGy,even more preferably an electron beam dose of 10 to 120 kGy, mostpreferably an electron beam dose of 15 kGy to 110 kGy.

The preferred embodiments of the method according invention also applyto the uses and immunogenic compositions and vaccines according to theinvention, as far as applicable.

FIGURES

FIG. 1: shows the PRRS virus activity after treatment with the electronbeam (gray) and without irradiation (black). Stated values are TCID50per ml of virus solution. Dose: 100 kGy.

FIG. 2: shows the results for the antigen integrity after treatment.PRRS viruses were treated with an electron beam (gray), formaldehyde(stripes) or untreated (black). The integrity of the antigens wasdetermined via incubation using a polyclonal serum from a PRRSV infectedpig. Stated values are OD (optical density) in the ELISA. Dose: 100 kGy.

FIG. 3: shows the results for the integrity of the virus envelope aftertreatment. PRRS viruses were treated with an electron beam (gray),peroxide (stripes) or untreated (black). The integrity of the virusenvelope was analyzed using an antibody against the capsid (N−) proteinof PRRSV. Stated values are OD in the ELISA. Dose: 100 kGy.

FIG. 4: H3N8 viruses were treated with low-energy electrons of 200 kGy(E) or 0 kGy (NC) and their activity measured by TCID50 determination.

FIG. 5: the integrity of the antigens was determined by incubation witha serum from an influenza-positive human. Values are absorption signalsin an ELISA test. E: H3N8 viruses treated with an electron beam dose of200 kGy; NC: H3N8 viruses treated with 0 kGy (control).

EXAMPLE 1 SUMMARY OF THE EXPERIMENTS

The experiments were carried out, for example, using the PRRS virus(porcine respiratory and reproductive failure syndrome virus). Thisvirus is a single-stranded, positive strand RNA virus of theArteriviridae family. The virus affects pigs and causes annual losses inthe pig industry in the billions.

PRRSV in 100 μL of liquid medium was conducted through the electron beamand irradiated with 100 kGy. The amount of virus used was 2*10⁴ TCID50per batch. Subsequently, the activity of the pathogens and theconservation of their antigens was investigated.

For the activity determination, the viruses (and the untreated controls)were added to Marc145 cells and the TCID50 value determined three dayslater. The irradiation resulted in a reduction of the activity by 2.5logarithmic steps compared to the control (FIG. 1). The quality of theantigens was investigated by measurements using antibodies. For thispurpose, sera from pigs which had been infected with a vaccine strain ofPRRSV were investigated. Immunisation with a live virus leads to acomprehensive humoral immune response against the antigens in theiroriginal undamaged state. Therefore, the extent of binding of polyclonalantibodies from an animal thus immunized to an antigen is a directindicator of the integrity of the antigen. FIG. 2 clearly shows that thebinding properties of the pig serum to the PRRSV viruses have hardlychanged as a result of irradiating the viruses. Consequently, almost allantigens are still in their original undamaged state, although the viruswas inactivated by 2.5 logarithmic steps. PRRS viruses were alsoinactivated with formaldehyde for comparison. This process resulted in asignificant decrease in the ELISA signal and therefore a cleardestruction of the antigens.

It was also investigated to what extent the inactivation process affectsthe virus structure. This was measured using an antibody against thecapsid protein (N-protein) of the PRRSV. This protein is protected bythe intact virus envelope and is not accessible by the antibody.Therefore, a signal indicates a damaged virus envelope. As FIG. 3 shows,the virus envelope remains intact after irradiation while the peroxideinactivation according to Amanna et al. (supra) leads to significantdamage to the structure. These data indicate that during the electronirradiation the inactivation is presumably due mainly to the destructionof the nucleic acids while the antigens and the virus structure remainlargely unaltered.

The method described, therefore, is suitable for preparing inactivatedvirus particles, e.g. for the preparation of vaccines, where it hasclear advantages over formaldehyde: the antigens are far betterpreserved and the addition of toxic chemicals can be dispensed with.

EXAMPLE 2: MATERIALS AND METHODS

Virus Culture, Inactivation and Irradiation

Cell cultures of Marc-145 cells were infected with PRRSV (DV vaccinestrain). After three days, the supernatants were removed and centrifugedat 4000×g at 4° C. for 15 minutes. The supernatants thus clarified werelayered on a 15% sucrose cushion and ultracentrifuged at 100 000×g forthree hours. This supernatant was removed and the pellet resuspended insterile PBS (phosphate-buffered saline, pH 7.4). After determining theinfectivity, the virus suspension was adjusted to a concentration of2*10⁵ TCID50/mL. Each 100 μL of this solution was added to 6-well plates(which had been previously coated with 0.5% agarose) and irradiated at50, 100 and 200 kGy. Negative controls were treated identically up tothe irradiation.

After irradiation, the virus-containing solution was removed and wasfurther used in TCID50 and antigen measurements. PRRSV was inactivatedwith 0.3% formaldehyde for 22 hours. The formaldehyde was then removedagain from the virus suspension by dialysis. The inactivation byperoxide was carried out according to Amanna et al. (supra) in 3% H₂O₂for 22 hours, followed by dialysis.

TCID50 Measurements

In order to investigate the activity of the irradiated viruses, serialdilutions (each in steps of 1:10) of the virus suspensions in Marc-145cells were added to 96-well plates. Three days later, the cytopathiceffect (CPE) was determined. The TCID50 corresponds to the dilution atwhich 50% of the infected cell culture wells still have a CPE.

Antigen Measurements

1.5 μL of the virus suspension (irradiated and control samples) wereincubated overnight at 4° C. in 96-well microtitre plates. The next day,an ELISA (enzyme-linked immunosorbent assay) was carried out accordingto a standard protocol. To detect the antigens, serum from aPRRSV-infected (DV vaccine strain) pig was used (dilution 1:100). Forthe detection, a secondary anti-pig IgG antibody was used (conjugatedwith horseradish peroxidase, Zymed) at a 1:5000 dilution.

Irradiation with Electrons

The composition comprising PRRSV viruses was thinly applied to a largeagarose surface for the irradiation. In detail, the following wascarried out:

1.) Preparation of 0.5% agarose gels in PBS,

2.) Pouring the gels into gel pouring apparatus for 1 mm layerthickness,

3.) Cutting out the gels in circular form of 3.5 cm diameter,

4.) Drying the gels (ca. 14 h under continuous sterile work bench) inpetri dishes (3.5 cm diameter), the dosimetry negative controls werethen dried with a dosimeter foil already inserted,

5.) Dispensing 100 μL of virus suspension (pure PBS for dosimetrynegative controls, ca 15 min. exposure)

6.) Packaging with PET/PE film,

7.) Irradiation under the conditions stated below.

The irradiation was carried out by quasi-stationary irradiation of 100ml of medium in each case in air.

A continuously operating electron beam emitter (Navarone type,manufacturer: COMET) was used. The electrons were accelerated to 150keV, the beam current was 5 mA. Distance in air between the compositioncomprising PRRSV viruses and the electron emission window: 45 mm.

The application of energy doses of 50, 100 and 200 kGy was performed insingle steps of 25 kGy each (corresponding to 2, 4 or 8 cycles of thesamples or linear passages each of 115 mm/sec). The target doses couldbe achieved under standard atmospheric pressure air with an accuracy ofca. 10%.

The documentation of the applied dose was performed spectrometricallyusing pararosaniline cyanide dosimeter films and the Risöscan system ata measuring wavelength of 554 nm. For irradiation at 100 kGy, thedosimeter film was changed after 50 kGy since the dosimeter typementioned has a measuring range of up to a maximum of 80 kGy. For thetarget dose of 200 kGy, the dosimeter film was correspondingly changedafter 50, 100 and 150 kGy.

The blank sample showed no dose input, i.e. the applied dose is dueexclusively to the electron beam treatment and the contact with PBS andthe agarose gels did not cause any change to the dosimeter strips.

EXAMPLE 3: IRRADIATION OF INFLUENZA VIRUSES

Influenza A viruses (equine influenza H3N8, strain A/equine2/Miami/1/63) were propagated in MDCK cells and concentrated byultracentrifugation analogously to PRRS viruses (Examples 1 and 2).

The irradiation with electron beams was also conducted analogously toExamples 1 and 2 but at a dose of 0 kGy (control) and 200 kGy.

The activity measurements were performed via a TCID50 endpointdetermination. The antigens were assayed in the ELISA format using theserum of an influenza A-infected human.

As with PRRSV, inactivation of the influenza viruses is shown. At a doseof 200 kGy, no active viruses were still detectable in the cell culture(FIG. 4). Nevertheless, antigens are still present and largely unchanged(FIG. 5).

1. A method for inactivating viruses, characterized in that animmunogenic composition or vaccine comprising at least one virus isirradiated with electron beams, said immunogenic composition or vaccinecomprising at least one virus (i) being liquid, in particular being asuspension, and (ii) comprising at least one viral immunogen.
 2. Themethod as claimed in claim 1, characterized in that the at least onevirus is selected from: (i) an enveloped virus or non-enveloped virus,and/or (ii) a dsDNA virus, dsRNA virus, ssRNA virus or ssDNA virus,and/or (iii) a human pathogenic and/or animal pathogenic virus, the atleast one virus preferably being selected from a human pathogenic and/oranimal pathogenic enveloped dsRNA virus, enveloped ss(−)RNA virus orenveloped ss(+)RNA virus, particularly preferably a) that the at leastone virus is selected from a human pathogenic and/or animal pathogenicss(+)RNA virus, very particularly preferably that the at least one virusis selected from a virus of the Arterividae family, and even morepreferably that the at least one virus is a porcine reproductive andrespiratory syndrome virus (PRRS virus), or b) that the at least onevirus is selected from an animal pathogenic virus, the influenza A and Bvirus, the TBEV virus, the IPV virus and the hepatitis A virus.
 3. Themethod as claimed in either of claim 1 or 2, characterized in that thevaccine or immunogenic composition (i) comprises one virus or (ii) twoor more different viruses.
 4. The method as claimed in any of claims 1to 3, characterized in that the electron beams are accelerated at lowenergy or moderate energy, preferably accelerated with an accelerationenergy of between 150 key and 700 keV, more preferably of between 200keV and 500 keV, even more preferably of between 250 keV and 400 keVand/or are applied essentially under standard atmospheric pressure,preferably wherein the standard atmospheric pressure is present asatmospheric oxygen, nitrogen or carbon dioxide gas.
 5. The method asclaimed in any of claims 1 to 4, characterized in that the immunogeniccomposition or vaccine comprising at least one virus is irradiated withan electron beam dose in the range of 50 kGy to 300 kGy, preferably 50kGy to 200 kGy, further preferably 50 kGy to 150 kGy, more preferably 50kGy to 120 kGy, even more preferably 50 kGy to 110 kGy.
 6. The method asclaimed in any of claims 1 to 5, characterized in that the activity ofthe at least one virus after irradiation, preferably measured as aTCID50 value, is less than 5%, preferably less than 1%, more preferablyless than 0.1% of the activity prior to irradiation, even morepreferably that no activity of the at least one virus is stilldetectable after irradiation.
 7. The method as claimed in any of claims1 to 6, characterized in that the at least one virus is an envelopedvirus.
 8. The method as claimed in any of claims 1 to 7, characterizedin that the antigen structure of the viruses of the immunogeniccomposition or vaccine is substantially retained after irradiation,preferably that the binding of a polyclonal serum directed against thenon-inactivated virus to the at least one virus of the irradiatedimmunogenic composition or vaccine is at least 40%, preferably at least70%, more preferably at least 80%, even more preferably at least 90% ofthe binding of the polyclonal serum to the at least one virus of theimmunogenic composition or vaccine prior to irradiation, in particularwherein the binding of the polyclonal serum to the at least one virus ofthe immunogenic composition or vaccine is determined by ELISA.
 9. Themethod as claimed in any of claims 1 to 8, characterized in that thevirus structure of the viruses is substantially retained afterirradiation.
 10. The method as claimed in any of claims 1 to 9,characterized in that (a) the irradiation is carried out using a devicefor generating electron beams, (i) which is operated preferablycontinuously or rapidly pulsed, and/or (ii) which provides electronspreferably according to the cold or hot cathode principle and/or (iii)which is embodied as an axial emitter (scanner) or linear broadbandemitter, and/or (iv) the electrons, after discharge through an emissionwindow of the evacuated generating chamber of the device, preferably areapplied to the immunogenic composition or vaccine, wherein theimmunogenic composition or vaccine is preferably in a container, and/or(v) the immunogenic composition or vaccine is preferably incorporatedstatically in the device, or is continuously transported through theelectron beam, and/or (b) the dose rate is in the range of 1 kGy/0.1 secto 150 kGy/1000 sec and/or (c) the irradiation time is between 0.1 secto 1000 sec, preferably between 1 and 100 sec, and/or (d) thetemperature of the immunogenic composition or vaccine prior toirradiation is between 1° C. and 40° C., preferably between 5° C. and37° C., more preferably between 10° C. and 32° C., even more preferablybetween 15° C. and 30° C., and/or (e) the temperature increase of theimmunogenic composition or vaccine after irradiation compared to beforeirradiation is between 1° C. and 15° C., preferably between 2° C. and10° C., and/or (f) the temperature of the immunogenic composition orvaccine after irradiation is between 2° C. and 41° C., preferablybetween 6° C. and 38° C., more preferably between 11° C. and 33° C.,even more preferably between 16° C. and 31° C., and/or (g) the densityof the immunogenic composition or vaccine is between 0.9 and 2 g/cm³,preferably between 1.0 and 1.8 g/cm³, and/or (h) the immunogeniccomposition or vaccine comprising at least one virus is a liquidsuspension comprising water, preferably is a suspension of the at leastone virus in an aqueous solution, wherein the aqueous solutionparticularly preferably comprises one or more buffer substances.
 11. Themethod as claimed in any of the preceding claims, characterized in thatthe immunogenic composition or vaccine comprises (a) one or moreadjuvants, and/or (b) pharmaceutically acceptable excipients and/orauxiliaries and/or (c) one or more further immunogens.
 12. The method asclaimed in claim 11, characterized in that one or more furtherimmunogens is selected from (a) an organic substance, particularly aprotein, which may be glycosylated or non-glycosylated, a nucleic acid,a toxin, or a sugar molecule which is optionally bound to a support, and(b) a virus or a living organism, particularly a bacterium, wherein thevirus or living organism may be active or inactivated.
 13. A method forpreparing a vaccine comprising at least one viral immunogen,particularly a vaccine comprising a viral whole particle vaccine,characterized in that: (a) the method is carried out as claimed in anyof claims 1 to 11, (b1) one or more adjuvants are optionally added tothe immunogenic composition comprising at least one virus, and/or (b2)one or more pharmaceutically acceptable excipients and/or auxiliariesare optionally added to the immunogenic composition comprising at leastone virus, and/or (b3) one or more further immunogens are optionallyadded to the immunogenic composition comprising at least one virus,wherein the steps (a) to (b3) are carried out in any sequence.
 14. Themethod for preparing a vaccine as claimed in claim 13, characterized inthat the following further steps are carried out: (c) sterilising theimmunogenic composition, and/or (d) filling the immunogenic compositionin a container, wherein steps (a) to (d) may be carried out in anysequence, and following steps (a) to (d) the vaccine is optionallydried, freeze-dried or frozen.
 15. An immunogenic composition orvaccine, preferably vaccine, particularly preferably comprising aninactivated viral whole particle vaccine, which may be prepared by amethod of claims 1 to
 14. 16. An immunogenic composition or vaccine,preferably vaccine, comprising an inactivated viral whole particlevaccine for an enveloped or non-enveloped virus, characterized in that(a) the activity of the virus in the immunogenic composition or vaccineis less than 10%, preferably less than 1%, more preferably less than0.1% of the activity of the same number of non-inactivated viruses, and(b) the antigen structure of the inactivated viruses in the immunogeniccomposition or vaccine is substantially the same compared to the samenumber of non-inactivated viruses.
 17. The immunogenic composition orvaccine as claimed in claim 16, (a) wherein the virus structure of theinactivated viruses is substantially the same compared to the samenumber of non-inactivated viruses, and/or (b) wherein the immunogeniccomposition or vaccine has been irradiated with an electron beam, and/or(c) wherein no activity of the at least one virus is detectable in theimmunogenic composition or vaccine.
 18. The immunogenic composition orvaccine as claimed in any of claims 15 to 17, wherein the immunogeniccomposition or vaccine has been irradiated with an electron beam dose inthe range of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, furtherpreferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, evenmore preferably 50 kGy to 110 kGy.
 19. The immunogenic composition,preferably vaccine, as claimed in any of claims 15 to 18, for use as avaccine, in particular for preventing or treating viral infections ordisorders caused by the virus.
 20. The use of electron beams (a) forinactivating viruses in an immunogenic composition or vaccine comprisingat least one virus, said immunogenic composition or vaccine (i) beingliquid, in particular being a suspension, and (ii) comprising at leastone viral immunogen, and/or (b) for preparing an inactivated viral wholeparticle vaccine.
 21. The use as claimed in claim 20, wherein theelectron beams are accelerated at low energy or moderate energy,preferably accelerated with an acceleration energy of between 150 keVand 700 keV, more preferably of between 200 keV and 500 keV, even morepreferably of between 250 keV and 400 keV and/or are applied essentiallyunder standard atmospheric pressure, wherein the standard atmosphericpressure is present as atmospheric oxygen, nitrogen or carbon dioxidegas and/or the electron beam dose is in the range of 50 kGy to 300 kGy,preferably 50 kGy to 200 kGy, further preferably 50 kGy to 150 kGy, morepreferably 50 kGy to 120 kGy, even more preferably 50 to 110 kGy. 22.The use of a device for generating electron beams (a) for inactivatingviruses in liquid immunogenic compositions, more preferably liquidvaccines, and/or (b) for preparing an inactivated viral whole particlevaccine.
 23. The use as claimed in claim 22, wherein the device a) issuitable for emitting electron beams accelerated at low energy ormoderate energy, preferably accelerated with an acceleration energy ofbetween 150 keV and 700 keV, more preferably of between 200 keV and 500keV, even more preferably are accelerated between 250 keV and 400 keVand/or for applying electron beams essentially under standardatmospheric pressure, and/or b) is suitable for delivering an electronbeam dose of 50 kGy to 300 kGy, preferably 50 kGy to 200 kGy, furtherpreferably 50 kGy to 150 kGy, more preferably 50 kGy to 120 kGy, evenmore preferably 50 to 110 kGy.
 24. The use of a method as claimed in anyof claims 1 to 14 for preparing an inactivated viral whole particlevaccine.